The international pharmaceutical market is under increasing pressure to develop new methods for the identification of novel therapeutics and drugs. Most diseases are complex, with multiple genes contributing to susceptibility, initiation, progression and modulation of the disease. Unlike diseases caused by single gene defects, the majority of human diseases are determined by the additive and synergistic interactions between gene products and the environment. Common diseases are, in effect, emergent properties of a complex system. It may not be specific biological elements that are flawed, but a combination of conditions that gives rise to a diseased state. Analyzing these emergent properties and elucidating the network of such interactions can make it possible to identify optimal, valid targets and drugs to treat common diseases.
The central dogma of biology describes the transfer of genetic information from DNA to RNA to protein, with the vast majority of cellular activity being controlled at the protein level. While enormous progress has been made in the areas of DNA and RNA analysis, protein analysis remains labor-intensive and difficult. Protein analysis is further complicated because of the diverse activities and characteristics of proteins. Protein expression levels, catalytic protein activities, protein modifications, protein-protein interactions, protein-nucleic acid interactions, and protein-small molecule interactions combine in a multitude of ways to yield a highly complex network of interactions within cells. Elucidation of the functions of many proteins, and the pathways and networks in which they interact, can lead to the discovery of the overall biological properties of a given system. These properties reveal themselves, or emerge, as one proceeds along the experimental path.
To fully analyze the properties of a complex system, it is generally necessary to perform complex biological experiments, involving thousands of samples (see, e.g., Houston et al. (1997) Curr. Opin. Biotechnol. 8:734–40). These experiments involve systematic perturbation of cellular systems and subsequent monitoring of hundreds of variables. Analysis of this information can be used to elucidate the emergent properties of the biological system and lead to a better understanding of the complex pathways involved in diseases. Systems currently available do not monitor the expression levels and functional state of hundreds of proteins within each experiment.
Current technologies for protein expression analysis can be placed into two categories: probing via immunodetection and direct visualization. Immunodetection methods, such as western blots and enzyme-linked immunoabsorbent assays (ELISAs), use antibodies to recognize and bind to a protein and produce a corresponding signal (see, e.g., Gonzalez et al. (1998) Curr. Opin. Biotechnol. 9:624–31; Sarubbi et al. (1996) Anal. Biochem. 237:70–5; Tijssen et al. (1991) Curr. Opin. Immunol. 3:233–237; and Woo et al. (1994) Clin. Lab. Med. 14:459–71). ELISA assays use antibodies produced by and harvested from a host that has been inoculated with an antigen, and are typically analyzed individually and occasionally in duplexes. Multiple antibodies can be used to map the physical structure of a protein.
In addition, a number of direct visualization technologies have been used to detect and monitor proteins. Two-dimensional gel systems are used for the large-scale analysis of complex mixtures of proteins. These systems are capable of analyzing up to 10,000 proteins in a single gel (see, e.g., Arnott et al. (1998) Anal. Biochem. 258:1–18; and Celis et al. (1999) Curr. Opin. Biotechnol. 10:16–21), but are laborious to produce and challenging to analyze.
Another direct analysis method involves the construction of protein/reporter gene conjugates. In this case, the expression of a protein is monitored by detecting a expression of a detectable reporter protein, such as a green fluorescence protein, whose encoding nucleic acid sequence is physically coupled to the gene encoding a protein of interest (see, e.g., Gonzalez et al. (1998) Curr. Opin. Biotechnol. 9:624–31; and Suto et al. (1997) J. Biomol. Screening 2:7–9). Reporter gene systems have been exploited in high throughput screening systems and provide information about the activity of a particular protein. There are issues related to the impact of the conjugates on gene. These reporter systems generally only allow one or two proteins to be monitored simultaneously and are limited to the cell types into which these constructs can be introduced.
Use of the different protein detection technologies listed above is limited to either measuring a few proteins in a large number of samples or measuring thousands of proteins in small numbers of samples. The need to monitor tens to hundreds of proteins, including their expression levels and functional state, in a high throughput fashion has yet to be fulfilled. Therefore, it is an object herein to fulfill this and other needs.